11 research outputs found
Humoral Activity of Cord Blood-Derived Stem/Progenitor Cells: Implications for Stem Cell-Based Adjuvant Therapy of Neurodegenerative Disorders
<div><p>Background</p><p>Stem/progenitor cells (SPCs) demonstrate neuro-regenerative potential that is dependent upon their humoral activity by producing various trophic factors regulating cell migration, growth, and differentiation. Herein, we compared the expression of neurotrophins (NTs) and their receptors in specific umbilical cord blood (UCB) SPC populations, including lineage-negative, CD34<sup>+</sup>, and CD133<sup>+</sup> cells, with that in unsorted, nucleated cells (NCs).</p><p>Methods and Results</p><p>The expression of NTs and their receptors was detected by QRT-PCR, western blotting, and immunofluorescent staining in UCB-derived SPC populations (i.e., NCs vs. lineage-negative, CD34<sup>+</sup>, and CD133<sup>+</sup> cells). To better characterize, global gene expression profiles of SPCs were determined using genome-wide RNA microarray technology. Furthermore, the intracellular production of crucial neuro-regenerative NTs (i.e., BDNF and NT-3) was assessed in NCs and lineage-negative cells after incubation for 24, 48, and 72 h in both serum and serum-free conditions. We discovered significantly higher expression of NTs and NT receptors at both the mRNA and protein level in lineage-negative, CD34<sup>+</sup>, and CD133<sup>+</sup> cells than in NCs. Global gene expression analysis revealed considerably higher expression of genes associated with the production and secretion of proteins, migration, proliferation, and differentiation in lineage-negative cells than in CD34<sup>+</sup> or CD133<sup>+</sup> cell populations. Notably, after short-term incubation under serum-free conditions, lineage-negative cells and NCs produced significantly higher amounts of BDNF and NT-3 than under steady-state conditions. Finally, conditioned medium (CM) from lineage-negative SPCs exerted a beneficial impact on neural cell survival and proliferation.</p><p>Conclusions</p><p>Collectively, our findings demonstrate that UCB-derived SPCs highly express NTs and their relevant receptors under steady-state conditions, NT expression is greater under stress-related conditions and that CM from SPCs favorable influence neural cell proliferation and survival. Understanding the mechanisms governing the characterization and humoral activity of subsets of SPCs may yield new therapeutic strategies that might be more effective in treating neurodegenerative disorders.</p></div
Global gene expression changes in human UCB SPCs (NCs, lineage-negative, CD34<sup>+</sup>, and CD133<sup>+</sup> cells).
<p>The heatmap represents the expression levels of highly overexpressed genes (Fold Change >4). Individual genes are assigned according to the Gene Ontology classification of specific biological processes listed on the left side of the graph. Each column comprises a set of horizontal lines, each representing a single gene. Levels of gene expression are indicated on a color scale, with yellow corresponding to the highest level of expression and blue corresponding to the lowest level. The range of expression rate of the analyzed genes is shown below the graph. The diagram shows large-scale alterations in gene expression between NCs, lineage-negative SPCs, CD34<sup>+</sup> cells, and CD133<sup>+</sup> cells.</p
Effects of lineage-negative-conditioned medium on SH-SY5Y cells.
<p>Cells were exposed to lineage-negative-CM for 24 h and compared with IMDM-treated cells (control). A. Effects of lineage-negative-CM on BrdU incorporation by SH-SY5Y cells measured by flow cytometry (representative flow cytometry plots). Gate 1, proliferating population indicated by BrdU and DAPI staining (left plot, cells treated with IMDM; right plot, cells treated with lineage-negative-CM). B. Proliferation of SH-SY5Y cells measured by flow cytometry with data expressed as the percentage of proliferating cells. Data are presented as the mean ± S.D. for three independent experiments. *P<0.05 vs. control. C. The expression of the proliferation marker Ki-67 in SH-SY5Y cells was detected by immunofluorescent microscopy. Cells were stained with a monoclonal Texas Red-conjugated anti-Ki-67 antibody. To visualize the cell membrane, cells were stained with an Alexa Fluor 647-conjugated wheat germ agglutinin (WGA). The nuclei were stained with DAPI. Cells were treated for 24 h with IMDM or lineage-negative-CM. All images were acquired by using a LSM700 confocal system (Carl Zeiss, Jena, Germany). D. Expression of Bcl-2 mRNA in SH-SY5Y cells after 24 h treatment of lineage-negative cell conditioned medium detected by QRT-PCR. Data are expressed as a percentage of the control (IMDM-treated cells). Data are presented as the mean ± S.D. for three independent experiments. *P<0.05 vs. control.</p
The primers used for real time QRT-PCR reactions.
<p>The primers used for real time QRT-PCR reactions.</p
Stress-related, serum-free conditions increases BDNF and NT-3 expression in UCB NCs and lineage-negative cells.
<p>(A) QRT-PCR shows changes in mRNA expression of BDNF and NT-3 in NCs after 24, 48, and 72 h in serum-free media. Relative mRNA expression levels are normalized against BMG. Data are shown as median, quartiles, interquartile range, minimum, and maximum for twelve independent experiments. *P<0.05 versus 0 h. (B) QRT-PCR shows changes in the mRNA expression of BDNF and NT-3 in lineage-negative cells after 24, 48, and 72 h in serum-free media. Relative mRNA expression levels are normalized to BMG levels. Data are shown as median, quartiles, interquartile range, minimum, and maximum for six independent experiments. *P<0.05 versus 0 h. (C) Representative western blot analysis of BDNF and NT-3 protein levels in NC lysates. (D) Representative western blot analysis of BDNF and NT-3 protein levels in lineage-negative cell lysates.</p
UCB SPC populations continuously express neurotrophin receptors.
<p>(A) NT receptor mRNA expression levels in UCB-derived nucleated cells, lineage-negative, CD34<sup>+</sup>, and CD133<sup>+</sup> cell populations. Relative mRNA expression values are normalized against BMG levels. Data are shown as median, quartiles, interquartile range, minimum, and maximum for six independent experiments. *P<0.05 versus NCs (the exact statistical difference is presented in the Results). (B) Representative western blot analysis of NT receptor protein levels in SPC lysates. (C) Immunofluorescent analysis of NT receptor expression on the surface of SPCs. Nuclei were visualized by DAPI staining. Pseudo-coloring was assigned to each stain as follows: anti-TrkA, anti-TrkB, and anti-TrkC – green, anti-p75NTR – red, and nuclei – blue. All images were captured using the Pathway Bioimager System (BD Biosciences, Rockville, MD, USA). Representative data are shown from three independent experiments.</p
Effect of hAM CCM on HUVECs migration assayed by scratch test.
<p>There are results of 160 measurements, 8 independent assays with 10 measurements for test and control each. Median values and (P25, P75) are shown (n = 8, p < 0.05). Detailed description of the assay is in Material and methods.</p
Growth factors in hAM CCM.
<p>Forty-one growth factors were quantitated by antibody array and the obtained values were combined into following growth factor families: EGF family (EGF-2, HB-EGF, EGF-R); FGF family (bFGF, FGF-4, FGF-6, FGF-7); Hematopoietic factors HF (MCSF, MCSF-R, SCF, SCF-R); IGF family (IGF-1, IGF-2, IGF-1SR); IGFBP family (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6); Neurotrophic factors NF (bNGF, GDNF, NT-3, NT-4); PDGF family (PDGF-AA, PDGF-AB, PDGF-BB, PDGF-Ra, PDGF-Rb); TGF family (TGF-α, TGF-β, TGF-β2, TGF-β3); Vasculogenic factors VF (PLGF, VEGF, VEGF-R3, VEGF-D, VEGF-R2); some growth factors are presented separately: AR; G-CSF; GM-CSF; HGF. Each growth factor fluorescence value (FV) was measured and calculated as described in Materials and methods. (n = 4) (p < 0.05).</p
The example of real time migration assay of stimulated and controlled HUVECs directly from the X-Celligence system.
<p>The assay was repeated seven times with similar result during 25h observation at 37<sup>°</sup>C. The difference between migration curves for cells in cultures with presence of hAM CCM and in control medium was significant. (p < 0.05).</p
Effect of hAM CCM on chemotaxy indeks of BM MNCs.
<p>The chemotaxy index (CI) after 2.5 h at 37<sup>°</sup>C incubation time was calculated by dividing the number of cells in lower chamber by the number of cells added to the upper chamber counted at the start of the test. Median values and interquartile range (P25, P75) are shown (n = 12, p < 0.05).</p